Breast cancer remains a serious public health problem. Aside from skin cancer, breast cancer is the most common form of cancer in women, with a lifetime incidence rate in the US population of approximately 13%. Breast cancer also remains one of the top ten causes of death for women in the US, and the second leading cause of cancer deaths in this population. Like all forms of cancer, breast cancer can be considered as a molecular reprogramming of the normal cell. Thus, understanding the gene regulatory networks that exist in breast cancer cells is of fundamental importance.
While mutations in BRCA1 or BRCA2 genes impart a very high risk for development of breast cancer, such mutations exist in the population at low frequency (and generally act as recessive cancer genes), and thus cannot account for the majority of breast cancers. Mutations in other genes, including PT53, PTEN, STK11, CDH1, also impart significantly increased risk of disease. However, even together with BRCA1 and BRCA2, these mutations may only account for 20% of familial disease. Thus, multiple additional genetic factors account for the observed disease incidence. In addition, the complexity of disease means that there can be additive and synergistic effects of changes in other mediators, even in the context of BRCA1 and BRCA2 mutations as discussed herein.
Applicant identified, using microarray analysis, the early changes in gene expression in precursor thymocytes as they traversed a developmental checkpoint-termed positive selection. These studies led to identification of a gene encoding a nuclear protein subsequently designated TOX (Thymocyte selection-associated HMG-box protein). This protein contains a single centrally-located DNA binding motif known as an HMG-box, named after that found in canonical HMGB proteins. The HMG-box now defines a superfamily of proteins (which have 47 family members located in the human genome) that, despite diverse functions, share some general characteristics of DNA binding. HMG-box domains, including TOX, fold into three α-helices that form a concave L-shaped structure that binds the minor groove of DNA. HMG-box proteins also bind distorted DNA structures and often can induce bending and unwinding of the DNA helix to fit the protein domain structure. Two general classes of HMG-box proteins have been identified based on their mode of binding to DNA; those that exhibit sequence-specific binding and those that bind DNA in a sequence-independent but structure-dependent fashion. The latter class of proteins includes the canonical HMGB proteins themselves, while the former include transcriptional regulators, such as LEF-1. Both kinds of proteins, however, play roles in regulating gene expression, often by inducing or stabilizing architectural changes in chromatin and facilitating nucleoprotein complex formation. HMG-box proteins may also augment other nuclear functions that benefit from architectural changes in DNA, including antigen receptor gene rearrangement and chromatin remodeling. By inspection of key residues in the HMG-box domain (TOX-box), TOX is almost certainly a member of the sequence-independent DNA-binding family. O′Flaherty E and Kaye J., TOX defines a conserved subfamily of HMG-box proteins. BMC Genomics. 2003; 4(1):13.
In this case, TOX may be targeted by recognizing structural features of chromatin or, alternatively, by binding to other proteins. The TOX-box also defines a subfamily of proteins that includes three additional members (TOX2, 3, and 4). Wilkinson B, et al., Nat Immunol. 2002; 3(3):272-80. Based on a high degree of conservation of the TOX-box sequence, all family members are predicted to be sequence-independent DNA-binding factors. Outside of the DNA-binding domains, the N-terminal domains of family members are the next most similar, and this domain has transactivation activity. The C-terminal domains of the family members are most distinct and there is reason to think that they may function as interaction domains. Yuan S H, et al., TOX3 regulates calcium-dependent transcription in neurons. Proc Natl Acad Sci USA. 2009; 106(8):2909-14. The C-terminal domain of TOX3 particularly stands out from the rest of the family, as it is highly glutamine-rich.
TOX expression is tissue- and stage-specific (although not T cell specific), with the greatest expression observed in the thymus and markedly reduced expression in peripheral lymphoid tissues. Wilkinson B, et al., 2002. Detailed expression of other TOX family members, however, has been less well characterized. TOX2 has been reported to be expressed in rat ovarian granulosa cells and mouse retina. As described herein, Applicant discovered that expression of Tox4 mRNA to be fairly widespread. Overall, it appears that despite some overlap in tissue expression, different TOX family members may play greater or lesser roles in specific tissues. Applicant further discovered that even in the mouse brain, where Tox and Tox3 mRNA are both expressed, they have non-overlapping patterns of expression.
Applicant characterized mice deficient in TOX and showed that this nuclear factor is required for development of a number of key aspects of the immune system including development of CD4 T cells, lymph nodes, and NK cells. Aliahmad P and Kaye J., Development of all CD4 T lineages requires nuclear factor TOX. J Exp Med. 2008; 205(1):245-56. Together the data indicate that TOX is a key regulator of precursor cell differentiation in various contexts, presumably by regulating gene expression (FIG. 3). These results make it likely that other TOX family members will also be found to play important regulatory roles in various cellular contexts. The biological function of other TOX-family members in vivo has not been characterized. Recently, however, expression of TOX3 has been reported to link calcium signaling to c-fos regulation in isolated neurons.